Oocyte cryopreservation is of great importance to the advancement of assisted reproductive medicine, maintenance of animal resources, and livestock management. However, the commonly used methods today for oocyte cryopreservation by either slow-freezing or conventional vitrification have inherent drawbacks. For example, the slow-freezing (freezing: the transition of liquid water into ice crystal) approach is associated with inevitable cell injury due to ice formation and slow- freezing induced cell dehydration. The unusually high CPA (cryoprotectant) concentration (4 - 7 M) required by the conventional vitrification (vitrification: the transition of liquid water into an amorphous, glassy state rather than ice crystal) method can result in significant metabolic and osmotic injury in living cells even in a short exposure time of only a few minutes. Presumably, it is these inherent drawbacks that are responsible for the dismal outcome of oocyte cryopreservation to date. The goal of the proposed research outlined in this R01 proposal is to develop new strategies for cell cryopreservation by microencapsulating the cells in alginate microcapsule to vitrify at a low-CPA (low and non- toxic amount of cryoprotectants, = 1.5 M) concentration. The proposed low-CPA vitrification approach combines all the advantages of the commonly used slow-freezing and conventional vitrification techniques today while avoiding all their shortcomings. Oocytes of the naturally bred (outbred) Peromyscus will be used as the biological model in this project so that the results obtained from the proposed studies can be more transferable to achieve low-CPA vitrification of oocytes of other naturally bred mammals including humans. In addition, Peromyscus embryos will be used as the benchmark biological model in this project to test the new approach in view of the fact that embryo cryopreservation has been successful in general. It is believed that the proposed research and the novel low-CPA vitrification approach will have a significant impact on the field of oocyte cryopreservation for assisted reproductive medicine, maintenance of animal resources, and livestock management. PUBLIC HEALTH RELEVANCE: Oocyte cryopreservation is of great importance to the advancement of assisted reproductive medicine. We propose to develop a novel technology to achieve much improved performance for oocyte cryopreservation. This research will have a significant impact on the preservation of future fertility of women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments such as extirpative surgery, radiation, and chemotherapy.